The vWF is a blood coagulation factor which is produced by vascular endothelial cells or bone marrow megakaryocytes and is present as a multimer structure (molecular weight 500 to 20,000 kDa) composed of subunits of a single type each having 2,050 amino-acid residue (monomer about 250 kDa) and bound by S—S bond. The blood concentration is about 10 μg/ml, and generally one with higher molecular weight has a higher specific activity.
The vWF has two major functions as a blood coagulation factor; one is a function as a career protein to bind to and stabilize blood coagulation factor VIII and the other is a function to allow blood platelets to adhere and aggregate to the tissue beneath vascular endothelial cells at damaged vascular wall and to form a platelet thrombus.
Thrombotic thrombocytopenic purpura (TTP) is a disease which forms platelet thrombus in body tissue arterioles and capillary vessels throughout the body, and the mortality related to this disease increased by about 3 times in 1971 through 1991 in spite of progress of recent medical technology. Pathologically, TTP is considered to be caused by disorder of vascular endothelial cells and platelet aggregation in blood vessels, and immunohistologically significant amount of vWF is observed in the formed platelet thrombus, and it is supposed that vWF plays an important role in the origin thereof. The multimer structure of vWF in the patient with TTP shows primacy of normal or high molecular vWF, and particularly, unusually large vWF multimer (ULvWFM) usually not observed and large vWF multimer (LvWFM) are presumed to play an important role in promotion of platelet aggregation and microthrombus formation under high shear stress. In the meantime, it has been known that vWF is decomposed at the position of 842Tyr-843Met by the action of vWF cleaving enzyme (vWF-cleaving protease) in the circulating blood in healthy people under high shear stress. Therefore, as for TTP, a scenario is assumed in which the activity of the above-mentioned enzyme in plasma is lowered by some cause, ULvWFM to LvWFM increase to enhance platelet aggregation, thereby causing platelet thrombus formation in the blood vessel.
The gene encoding vWF cleaving enzyme which is the main part of the activity having this enzyme activity, also known as ADAMTS-13, was cloned by the applicant of this application in 2001 (WO 02/088366). The knowledge about the molecular structure of ADAMTS-13 is summarized below.
As for the domain composition of ADAMTS-13, propeptide is present following the signal peptide and subsequently, RQRR (SEQ ID NO: 21) sequence of cleavage motif of Furin is present and a metalloprotease domain which contains a reprolysin type zinc chelate region consisting of a consensus sequence of HEXXHXXGXXHD (SEQ ID NO: 22) follows (to P285stop). And via a disintegrin-like domain which is found in snake venom metalloprotease (to W387stop), it connects to the first Tsp1 motif (Tsp1-1) consisting of about 50 to 60 residues which is generally considered to be important for molecule recognition (to Q449stop) and further continues to Gys-rich region in which RGDS (SEQ ID NO: 23) sequence, one of cell adhesion motifs, is included (to T581stop). Subsequently, via a spacer domain which consists of about 130 amino acid residues containing no cysteine residue (to W688stop), additional seven Tsp1 motifs (Tsp1-2 to 8) follows again, and CUB domain-1 and -2 continue which are known to be first found in complement component C1r or C1s.
In the meantime, there has not been established an efficient and high-purity purification process for this enzyme nor a method for qualitative and/or quantitive diagnosis on the amount of existence of this enzyme. Furthermore, no method for diagnosing an autoantibody-positive patient to this enzyme has been established, and the domain essential for expression of the activity of this enzyme has not been identified.